Fabrication of pure Bi2WO6 and Bi2WO6/MWCNTs nanocomposite as potential antibacterial and anticancer agents

An essential research area for scientists is the development of high-performing, inexpensive, non-toxic antibacterial materials that prevent the transfer of bacteria. In this study, pure Bi2WO6 and Bi2WO6/MWCNTs nanocomposite were prepared by hydrothermal method. A series of characterization results by using XRD FTIR, Raman, FESEM, TEM, and EDS analyses, reveal the formation of orthorhombic nanoflakes Bi2WO6 by the addition of NaOH and pH adjustment to 7. Compared to pure Bi2WO6, the Bi2WO6/MWCNTs nanocomposite exhibited that CNTs are efficiently embedded into the structure of Bi2WO6 which results in charge transfer between metal ion electrons and the conduction or valence band of Bi2WO6 and MWCNTs and result in shifting to longer wavelength as shown in UV–visible and PL. The results confirmed that MWCNTs are stuck to the surface of the microflowers, and some of them embedded inside the Bi2WO6 nanoflakes without affecting the structure of Bi2WO6 nanoflakes as demonstrated by TEM. In addition, Pure Bi2WO6 and the Bi2WO6/MWCNTs nanocomposite were tested against P. mirabilis and S. mutans., confirming the effect of addition MWCNTs materials had better antibacterial activity in opposition to both bacterial strains than pure Bi2WO6. Besides, pure Bi2WO6 and the Bi2WO6/MWCNTs nanocomposite tested for cytotoxicity against lung MTT test on Hep-G2 liver cancer cells, and flow-cytometry. Results indicated that pure Bi2WO6 and the Bi2WO6/MWCNTs nanocomposite have significant anti-cancer efficacy against Hep-G2 cells in vitro. In addition, the findings demonstrated that Bi2WO6 and Bi2WO6/MWCNTs triggered cell death via increasing ROS. Based on these findings, it appears that pure Bi2WO6 and the Bi2WO6/MWCNTs nanocomposite have the potential to be developed as nanotherapeutics for the treatment of bacterial infections, and liver cancer.

The photocatalyst semiconductors have garnered increasing interest recently as an antibacterial agent due to considerable chemical stability and broad-spectrum antimicrobial capabilities.The antibacterial effects of photocatalysts semiconductors on both negative and positive bacteria have been studied in many research [1][2][3] .Among a typical Aurivillius oxide, Bismuth tungstate (Bi 2 WO 6 ) is a widely utilized Bi-based semiconductor due to its exceptional fundamental physical and chemical properties 4 .Bi 2 WO 6 is a favorable photocatalyst with a narrow visible light absorption band (2.8 eV) for environmental treatment fields, friendly characteristics, chemically stable, surface Plasmon response (SPR), and low toxicity.It has also been demonstrated that Bi 2 WO 6 exhibited photocatalytic degradation of organic pollutants and good potential for applications as an antibacterial agent [5][6][7] .In terms of controllable synthesis, Bi 2 WO 6 nanoplates, nanosheets, nanorods, and nanoflowers can be made using the hydrothermal procedure, sol-gel technique, calcination, and electrodeposition.In addition, the hydrothermal procedure of Bi 2 WO 6 frequently results in the creation of 2D nanostructure plates along the (001) plane as revealed in the previous papers [8][9][10][11][12][13] .Moreover, depending on how the experimental circumstances are adjusted,

Synthesis of pure Bi 2 WO 6 and Bi 2 WO 6 / MWCNTs nanocomposite
Pure Bi 2 WO 6 was formed by the hydrothermal technique.To begin, 80 mL of D.W. was dissolved with 2 mmol of Bi(NO 3 ) 3 .5H 2 O, 1 mmol of Na 2 WO 4 .2H 2 O, and 0.05g of [CTAB].The final solution was stirred using a magnetic stirrer at room temperature for 30 min and adjusted to pH 7. Then the white suspension solution was sealed into a 100 mL autoclave of Teflon lined.After being sealed, the autoclave was maintained for 16 h at 180 °C in an oven with convection.Then the autoclave was cooled to ambient temperature and the white precipitate was developed.Bi 2 WO 6 was washed several times in distilled water and ethanol before being dried in an oven with convection for 10 h at 60 °C.The same process was utilized to form the Bi 2 WO 6 /MWCNTs nanocomposite.0.4 g of functionalizing F-MWCNTs were added after 0.4 g of prepared Bi 2 WO 6 had been dissolved in 50 mL of deionized water and stirred for one hour in a beaker using a magnetic stirrer.Then, to accomplish dispersion, the mixture was dissolved in 100 mL of deionized water using ultrasonication for two hours.After an hour, the solutions are mixed and stirred.While this was going on, 2 mL of sodium hydroxide (NaOH) with a concentration of 4 mol/L was thoroughly dissolved in distilled water by a magnetic stirred at 25 °C for 20 min.Moreover, the Bi 2 WO 6 /MWCNTs solution was continuously stirred as the (NaOH) solution was added dropwise (3 drops per minute) until the pH was adjusted to 7. The combination was placed in a stainless steel autoclave with a Teflon liner that held 150 mL and heated for 16 h at 180 °C.After that, the autoclave was cooled to room temperature.

Characterization
Using XRD diffractometer (XRD diffraction 6000, Shimadzu) and CuK radiation (λ = 1.542Å), current 30 mA and voltage 40kV.The structure of pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite samples were investigated.The recorded data fell between 10° and 60°.The average particle size (D) was estimated using Scherrer equation D = kλ/βcosθ, where k is a constant (0.89), β is the full-width half maximum (FWHM) and θ is Bragg law.FTIR spectrum (8400S, Shimadzu) was employed to analyze the boundary construction of the resulting samples.The samples were mixed with KBr powder and pressed to form the semitransparent pellets.Using a wavelength of about 250 nm, the Photoluminescence (PL) spectroscopy was carried out using (Cary Eclipse fluorescence model, Iran).The optical properties were performed using UV-visible spectroscopy (Shimadzu UV-1800 spectrophotometer) and using suspension with a concentration of 0.1mg/mL in DW at a wavelength ranging from 200 to 800 nm at room temperature.The Raman spectra of Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite were achieved in a range of (200-1800) nm (Raman spectroscopy, Takram N1-541).The samples absorbed laser light photons (λ = 633 nm as excitation source), which are then reemitted.The field emission scanning electron microscopy (FE-SEM, TESCAN, MIRA3) images of Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs samples were captured to observe their morphology.The sample reflects the electrons that are emitted from the filament, and either secondary electrons or backscattered electrons are used to create pictures.Furthermore, Energy-dispersive X-ray spectroscopy (EDS) was used to determine the composition elements of the samples.For more details to study the morphology of samples, transmission electron microscopy (TEM, Philips-EM-208S) was used.High energy electron beam transmitted was used through a very thin sample to image and analyze the nanostructures and nanocomposite materials with atomic-scale resolution.Besides scanning electron microscopy (SEM Apreo2, Thermo Fisher Scientific, USA) was carried out to morphology the performance of Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs products on bacterial cells.Besides, energy-dispersive X-ray spectroscopy (EDS) was used as a relatively simple but powerful technique for the elemental analysis of samples.

Antibacterial activity assay
The antibacterial activity of Pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite was assessed by agar well diffusion assay against P. mirabilis as gram's negative and S. mutans as gram's positive bacterial strains.Initially, about 20 mL of Muller-Hinton (MH) agar was aseptically poured into sterile Petri dishes.The bacterial species were collected from their stock cultures using a sterile wire loop.After culturing the organisms, using the tips of a sterile micropipette, 6 mm-diameter wells were bored on the agar plates.Different concentrations (62.5, 125, 250 μg/mL) of the bacterial samples were cultivated at 37°C overnight on Muller-Hinton (MH) agar (HiMedia India) comprising pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite.The agar plates were photographed, and the inhibition zones were measured.

Scanning electron microscopy (SEM) of bacteria permeability
The damages to the outer cell membrane permeability were recognized by monitoring the bacterial morphology on agar plates.As explained in the above section, pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite samples are taken in different concentrations.Bacterial cells of P. mirabilis as Gram's negative and S. mutans treated with pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite were centrifuged at 4000 rpm and washed three times using phosphate buffer solution (50 mM, pH = 7.3) as well as using untreated (control).Then thin smear of the suspension was spread on the glass slide and maintained at room temperature until dry.Slides samples were visualized with a Scanning Electron Microscopy (SEM).

Crystal violet staining
P. mirabilis and S. mutans at concentration (1 × 10 6 CFU/mL) were grown in 24-well plates and treated with pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite at concentrations 125 μg/mL for 24h.After that, the samples were washed with PBS and P. mirabilis and S. mutans adhered wells were stained with crystal violet (0.1%, Sigma, USA) after rinsing twice with D.W. To measure biofilm development, 0.2 mL of 95% ethanol was added to crystal violet stained wells and incubated for 2 h while being shaken.The optical density was then calculated at 595 nm.

Investigation of bacterial biofilm metabolic activity
Biofilms were formed in glass tubes in the presence and absence of the pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs samples 35 .After incubation at anaerobic conditions at 37 °C and for 48 h, the biofilm suspension was stained with a Live/Dead stain Kit and analyzed by flow cytometry.Briefly, 10 μL of Syto9 (30 μM) was added for 10 min, and then, 10 μL of propidium iodide (500 μM) was added for 10 min; the samples were washed 2 times in PBS and centrifuged for 2 min at 2000 rpm.The sample with two stain components was excited at 488 nm, and the emission was registered using the FITC channel for Syto 9 (530/30) and, (670/LP) channel for propidium iodide.The results of biofilm cell viability were expressed in the percentage of untreated control cells.

Cytotoxicity assay (MTT assay)
The cytotoxicity of the pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite was investigated by MTT assay.After cultivation overnight, Hep-G2 and REF cells were seeded into 96-well plates at a density of 1 × 10 www.nature.com/scientificreports/Following a wash with PBS, the cells were subjected to a three-hour treatment with an MTT solution containing 2 mg/mL (Invitrogen, Carlsbad, CA).After that, the solution was drained out of each well, and then 100 µL of DMSO was added to each one.A microplate reader was utilized to determine each sample's absorbance at a wavelength of 492 nm 37 .The equation that was used to determine the rate of inhibition of cell growth, also known as the percentage of cytotoxicity, is as follows.
where A represents the optical density of the control and B represents the optical density of the samples 38 .
Acridine orange/ethidium bromide staining (AO/EtBr) In 12-well plates, the Hep-G2 cells were collected and plated.Following a 24-h incubation period, the cells were exposed to Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite for 24 h.Following that, the cells were stained by10 µg/mL AO/EtBr for 2 min at 37 °C, and detected by the fluorescent microscope.

Flow cytometry assay
To measure the production of ROS in cells, a flow cytometry test was utilized.Hep-G2 cells were seeded at a density of 1 × 10 6 per well.Following overnight incubation, the cells were treated with Bi 2 WO 6 and Bi 2 WO 6 / MWCNTs nanocomposite for 8 h.After that, a ROS probe (DCFH-DA) at a concentration of 15 µM was added to the new medium and incubated for another 30 min in the dark.The fluorescence intensity of the cells was measured using a flow cytometer.In addition, flow cytometry assay was used to measure mitochondrial dysfunction assay using a Rhodamine probe, and Mitochondrial Membrane potential using (JC-1) probe.The fluorescence intensity of the cells was measured according to the manufactured protocol.

Apoptosis detection annexin V/PI assay
Cell apoptosis was investigated using a flow cytometry assay.Liver cancer cells (Hep-G2) were treated with Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite at a concentration (25 µg/mL).The cells were taken out and collected after 24 h.Cells were twice washed with cold PBS, and stained for 30 min with an Annexin V FITC and PI solution.The labeled cells were then evaluated using a flow cytometry assay.

Statistical analysis
The resulting data are the outcomes of three independent experiments.Data are represented as mean ± SD.Graph-Pad Prism (7) was used to carry out the statistical analysis via the application of the one-way ANOVA analysis of variance.The difference between means was assessed by LSD, in which p ≤ 0.05 was considered significant.*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 39 .

Results and discussion
Structural, optical, and morphological studies  1.In addition, the high intensity of the (131) and (200) peaks in Fig. 1 were related to the increase in the energy of the superficies of Bi 2 WO 6 , which enhances the quality of the crystallinity by reducing the defects in the Bi 2 WO 6 40 .In the state of Bi 2 WO 6 /MWCNTs nanocomposite, Fig. 1 (blue line), represented the characteristic peaks of the Bi 2 WO 6 /MWCNTs nanocomposite at 2θ = 28.30°,32.91°, 47.2°, 55.89°, 58.6° and 78.53° related to the (131), ( 200), ( 202), (133), and (262) planes of the orthorhombic structure of Bi 2 WO 6 , suggestion that the phase of Bi 2 WO 6 in Bi 2 WO 6 /MWCNTs nanocomposite has no significant change related to the addition of MWCNTs.However, the addition of low content of MWCNTs (less than 1% wt) may be responsible for the absence of MWCNTs peaks in the synthesized nanocomposite and their relatively lower crystallinity than pure Bi 2 WO 6 41 .Moreover, the value of the average crystallite size increases due to decreasing the β (the half-peak width becomes narrower) as listed in Table 1.The small amount of MWCNTs inclusion in the nanocomposite was said to have prevented MWCNTs from having an impact on the characteristic diffraction 42 .Additionally, it was discovered that the influence of additional MWCNTs caused the peak intensity to gradually decrease in comparison to the intensity in the pure Bi 2 WO 6 .
The FTIR spectra of pure Bi 2 WO 6 in the 500-4000 cm −1 wavenumber region are shown in Fig. 2a,b at room temperature.The strong bands between 500 and 1300 cm −1 were associated with the W-O, Bi-O, and W-O-W Table 1.Crystalline size of Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite by using hydrothermal method.

Materials θ (°) Hkl FWHM (°) Grain size (nm)
Pure Bi 2 WO 6  www.nature.com/scientificreports/stretching vibrations, as illustrated in Fig. 2a 43 .In particular, the W-O bending vibrations mode is corresponding to peak 731 cm −1 .The Bi-O stretching and deformation vibrations at peak 436 cm −144 , and the W-O-W stretching at 845 cm −145,46 , which match earlier papers [47][48][49] .The stretching vibrations of the -OH groups of adsorbed the H 2 O on the surface of the Bi 2 WO 6 sample were responsible for the broad absorption band at 3455 cm −1 .The O-H bond's bending vibration in the sample that was adsorbing water molecules was indicated by the vibration peak at 1624 cm −150 .The peaks at 2921 cm −1 and 2854 cm −1 were related to added surfactant-like methyl and methylene groups of CTAB which are consistent with previous papers 51,52 .Furthermore, Fig. 2b displayed the FTIR analysis of the Bi 2 WO 6 /MWCNTs nanocomposite, which exhibited a high and broad peak at 3460 cm −1 and a comparatively weak peak at 1622 cm −1 , respectively.These peaks are ascribed to the O-H stretching vibrations of the water molecules that have been adsorbed on the Bi 2 WO 6 and MWCNTs 53,54 .After the addition of MWCNTs, the peak at 1622 cm −1 becomes weaker, showing that partial surface O-H groups are ingested by the MWCNTs decoration 15 .The bond lengthening or weakening that causes the O-H peak at 3460 cm −1 to move to higher wave numbers is caused by an increase in the force constant.This phenomenon happens when the bond length becomes shorter.The modification in the surrounding atom's electronegativity may be the cause of the changing bond length 25 .The main absorption peaks of Bi 2 WO 6 were observed at (400-800) cm −1 which was assigned to the symmetric and asymmetric vibration of W-O at 725 cm −1 and the stretching vibration of Bi-O at 584 cm −1 , respectively 47 .Additionally, the peak of MWCNTs at 1224 cm −1 and 1052 cm −1 correspond to the C-O-C stretching vibration and the C-O stretching vibration, respectively [55][56][57] .As indicated in Table 2, the composition of Bi 2 WO 6 and the presence of a low quantity of MWCNTs were obvious.
The Raman spectra of Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite are shown in Fig. 3, respectively.The Raman peak positions of pure Bi 2 WO 6 in the wavenumber range of 200-1000 cm −1 correspond to the orthogonal Bi 2 WO 6 as shown in Fig. 3 (blue line) 58 .These bands can be related to the structure of W-O and Bi-O stretches being visible in the range of (700-800) cm −1 .Besides, the peak at 703 cm −1 exhibits a splatted pattern due to the distortion in W-O interaction owing to size reduction, and the peak at 787 cm −1 was related to Bi-O stretches 59 .The band at 295 cm −1 could be assigned to translational modes involving simultaneous motions of Bi +3 and WO 4 as a consequence of the addition of CTAB 60 .Similarly, vibrations in the region between (100-450) cm −1 for Bi 2 WO 6 are mainly attributed to rocking or bending modes of the WO 6 octahedron and BiO 6 polyhedron.The Bi-O peak at 297 cm −1 was split into more prominent bands as a consequence addition of CTAB.Furthermore, it is observed that CTAB prompts a reduction in the thickness and disordered Bi 2 WO 6 morphological www.nature.com/scientificreports/nanostructure.Therefore, it was suggested that the addition of CTAB is responsible for structural alternation that led to forming WO 6 octahedron 49,61 .Moreover, the Raman spectra obtained agree with the XRD results 62,63 .Figure 3 (red line) displays Raman spectra of Bi 2 WO 6 /MWCNTs nanocomposite.The peak at 703 cm −1 was related to W-O interaction and the peak at 787 cm −1 belonged to Bi-O stretches 64 .Similarly, to pure Bi 2 WO 6 , the bands relating to Bi rocking and transverse Bi 3+ motions appeared in the range of (200-400) cm −165 .When the amount of MWCNTs atoms were embedded into Bi 2 WO 6 , the intensity of the peak at 783 cm −1 was reduced compared to pure Bi 2 WO 6 .It might be attributed that some MWCNTs atoms were embedded in the O-W-O lattice and replaced some oxygen atoms that were originally bonded with W. In addition, the two weak peaks around 1595 cm −1 and 1346 cm −1 for Bi 2 WO 6 /MWCNTs nanocomposite are related to the D band and G band, respectively and the calculated D/G intensity ratio is about 1.1.The D band and G band of F-MWCNTs were about 1332 cm −1 and 1571 cm −1 , respectively as shown in Table 3.The D/G intensity ratio of Bi 2 WO 6 /MWCNTs increased compared with the intensity ratio of MWCNTs (D/G = 0.84).This shifting in intensity ratio towards high value due to the chemical oxidation and increasing D-band intensity was attributed to sp 3 bonds in F-MWCNTs after the hydrothermal method.Besides, the Raman spectral results reveal that MWCNTs are successfully introduced into the Bi 2 WO 6 lattice, particularly for ordered and disordered crystal structures of multi-walled carbon nanotubes 58,65 .In Fig. 4a,b, the UV-Vis absorption spectra of pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite synthesized by hydrothermal method are displayed, respectively.The UV-Vis absorption spectrum of pure Bi 2 WO 6 was studied in range (300-800 nm). Figure 4a showed a strong and broad absorption band in the range from the UV region 250 nm to the visible region 450 nm with a strong red shift edge at 423 nm.It can be seen that the Bi 2 WO 6 behaves as a crisp photo-absorption edge in the visible region, showing that the absorption appropriate to the band gap is caused by the intrinsic transition of the nanostructure rather than the transition from impurity levels to give the absorption edge energy which corresponds to E g = 2.7 eV in the set image.Furthermore, the shrinking band gap of pure Bi 2 WO 6 is always a result of the wavelength's redshift.In the state of Bi 2 WO 6 /MWCNTs nanocomposite as shown in Fig. 4b, It is found that the band gap of E g = 2.6 eV corresponds to a light absorption edge of around 465 nm for Bi 2 WO 6 /MWCNTs nanocomposite.Figure 4b absorption spectra make it clear that the Bi 2 WO 6 /MWCNTs nanocomposite is shifted to a longer wavelength (i.e.redshift) due to the high visible light  www.nature.com/scientificreports/absorption that occurs after the addition of a small amount of MWCNTs, which exhibit a clear redshift.Table 4 summarizes the observed band gaps for pure Bi 2 WO 6 and the Bi 2 WO 6 /MWCNTs nanocomposite.Furthermore, after the inclusion of MWCNTs, the energy gap lowers dramatically.This is because of the quantum effect which results in the band gap shrinking and shifts in the absorbance from the valance band to the conduction band towards a long wavelength 64 .Also, at the bottom of the conduction band, new localized levels are formed, and these levels are ready to receive electrons and generate tails in the optical energy gap, and these tails act to reduce the energy gap 66 .Since, a small amount of MWCNTs can alter the light absorption of nanocomposite, so confirming sample purity is always necessary when observing optical changes.
These results also agree with XRD patterns analysis related to the creation of oxygen vacancies and low intensity of nanocomposite during hydrothermal method with the addition of a small number of MWCNTs that can contribute to changes in optical properties of reduction band gap and reduced PL of nanocomposite as reveal in next paragraph.
The photoluminescence (PL) emission spectra resulted in Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite samples were used to investigate the charge carrier transfer efficiency and recombination rate of the photogenerated electron-hole (e-h) pairs as well as measurement was carried out to investigate the effect of MWCNTs on the photocatalytic process of semiconductor materials 67 .In Fig. 5, the PL spectrum of each sample had three relatively obvious peaks under the excitation wavelength of 250 nm.As shown in Fig. 5, high emission PL intensity means the rapid charge recombination rate of pure Bi 2 WO 6 semiconductor photocatalyst and is higher than that of charge carriers of Bi 2 WO 6 /MWCNTs nanocomposite 49,68,69 .Bi 2 WO 6 /MWCNTs reveals low PL intensity refers to a low rate recombination of electron-hole pairs as shown in Fig. 5.These results improved the separation rates of generated (e-h) pairs of nanocomposite and suggest that the addition of MWCNTs can effectively inhibit the recombination rates of electrons and holes.Besides, the results from UV-visible and PL analysis reveal the spectral changes in optical properties result from interfacial electronic interactions, modification of energy bands, and creation of interface transition phase between Bi 2 WO 6 and MWCNTs providing pathways for charge transfer and separation.The nanocomposite architecture enables synergistic coupling to enhance visible light.
As demonstrated in Fig. 6a,b, the morphology of synthesized pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite were examined using FESEM analysis, respectively.An expanded view in FESEM revealed that each particle has a flower-like structure made up of a collection of 2D flakes that radiate in all directions as shown in Fig. 6a 70 .Moreover, the high-magnified FESEM image in Fig. 6b, shows that pure Bi 2 WO 6 was made up of flower-shaped particles with a diameter of 1-2 μm.Moreover, the magnified FESEM in Fig. 6b reveals the crystallization of orthorhombic pure Bi 2 WO 6 .The microstructure of the crystalline nanoparticles then developed into a flower-like shape 71 .Larger particles increased at the expense of the lesser ones, by the Gibbs-Thomson law 72 .Besides, the average particle size distribution of pure Bi 2 WO 6 nanostructure by using image J was approximately about 1.09 μm as shown in the inset image [73][74][75][76] .
Furthermore, the particle size determined by FESEM images exceeds 10 times the particle size predicted by XRD.The layered structure of the Bi 2 WO 6 nanoparticles would be primarily responsible for the extreme www.nature.com/scientificreports/variation 77 .Furthermore, under hydrothermal conditions to yield Bi 2 WO 6 nanoflakes structures, nucleation processes driven by minimization of the total energy of the system lead to relatively high surface areas with larger sizes 61,77 .Additionally, the FESEM characterization of Bi 2 WO 6 /MWCNTs nanocomposite reveals a structure similar to the pure flower-like microstructures Bi 2 WO 6 as shown in Fig. 7a,b.The results showed that the inclusion of a small amount of MWCNTs has a straightforward impact on the morphology of composite made of Bi 2 WO 6 nanoflakes that have clumped together in Fig. 7b.This finding shows that nanocomposite fabrication is a useful strategy for preventing electron-hole pair recombination 48 .It is revealed that there are nanotubes are stuck to the surface of the micro flowers, and some of them embedded inside the Bi 2 WO 6 nanoflakes without affecting the structure of Bi 2 WO 6 nanoflakes and firmly suggesting the existence of MWCNTs in the Bi 2 WO 6 /MWCNTs nanocomposite formed.Moreover, when MWCNTs were added to the pure flower Bi 2 WO 6 , the morphology of the nanocomposite slightly changed which showed that the Bi 2 WO 6 /MWCNTs nanocomposite is made up of nanosheets as shown in Fig. 7b.The FESEM of Bi 2 WO 6 /MWCNTs nanocomposite was exhibited with particle sizes of about 0.997 μm as shown in the inset image of the size distribution histogram.The results reveal that the average particle size of nanocomposite was decreased compared with pure Bi 2 WO 6 due to F-MWCNTs exhibiting a considerable number of defects and an improved interaction with Bi 2 WO 6 .Additionally, the Bi 2 WO 6 acts  More detailed information on the morphology and microstructure of pure Bi 2 WO 6 nanostructure and Bi 2 WO 6 /MWCNTs nanocomposite was obtained by using TEM at different magnifications in Figs.9a-d and  As seen in Fig. 9c,d, the magnified image showing the edge indicates that the microsphere was constructed from nanosheets that ranged in size from 100 to 200 nm.Additionally, the nanoplates have a thickness of 20-30 nm [62][63][64][65] .The results obtained demonstrate the effective manufacture of pure Bi 2 WO 6 , which is consistent with the XRD analysis.
In the state of synthesized Bi 2 WO 6 /MWCNTs, as shown in Fig. 10a-d, it can be seen that the nanocomposite is composed of typical curly and flimsy nanoplates.Besides, a small amount of nanotubes is embedded inside the Bi 2 WO 6 nanoflake without affecting the structure.The majority of the area occupying the outer surface is the Bi 2 WO 6 layer, which becomes irregular after crystallization and occupies most of the space, the results are consistent with the FESEM analysis, which indicates that a low amount of MWCNTs have a small obvious influence on the morphology of Bi 2 WO 6 composites.It can be observed from Fig. 10c,d at high magnification, that the MWCNTs have been embedded inside the surface of Bi 2 WO 6 successfully.The surface area of the Bi 2 WO 6 / MWCNTs nanocomposite has increased comparatively due to the relatively small sizes of the nanostructure, which can improve the anticipated electrochemical performance.As the first essential step in the success of any functionalization procedure for the carbon nanotube surface is the treatment (oxidation) of the MWCNTs.Nevertheless, the increase in grain size may be attributed to the greater mobility of incoming reactants and surface diffusion which results in larger crystallites 66 .The above results demonstrate that the Bi 2 WO 6 /MWC-NTs nanocomposites are successfully formed indicating that Bi 2 WO 6 is essentially interacting with MWCNTs nanoparticles 67,68 .

Antibacterial activity of Pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite
Agar well diffusion assay is used as indicated in Fig. 11a,b to show the antibacterial impact of pure Bi 2 WO 6 nanostructure against P. mirabilis, a Gram-negative bacterial strain, and S. mutans, a Gram-positive bacterial strain.The agar well diffusion assay was performed at various test concentrations (26.5, 125, 250) μg/mL and the bacterial strains were cultured at 37 °C overnight.The inhibition zones against P. mirabilis were (17 ± 0.85, 19 ± 0.95, and 22 ± 1.1) mm, and against S. mutans, were (18 ± 0.9, 20 ± 1, 22 ± 1.1) mm, as demonstrated in Because their cell walls are more complex, gram-negative bacteria seem to be more resistant than gram-positive bacteria.The cell walls of S. mutans bacteria are distinct from those of P. mirabilis bacteria.There is only the peptidoglycan (PG) layer on the surface of S. mutans bacteria.On the other hand, P. mirabilis bacteria have cell walls with outside polysaccharide barriers that protect all polysaccharide structures from degradation 48 and whose outer surfaces are often resistant to hydrophobic compounds like NPs 69 .Additionally, one of the most crucial elements supporting the antibacterial effect of manufactured Bi 2 WO 6 nanostructure is their size, which increases their surface area by decreasing their size and enhances their contact with pathogens 70 .The crystallite structure of Bi 2 WO 6 layered can generate e-h pairs.As mentioned above, these species interact with H 2 O to produce functional groups like OH, H + , and O 2 that can affect the cell surface to break down various parts of the bacterial cell membrane.The loss of function brought on by cell rupture may finally result in cell death in the bacterium 71,72 .Furthermore, the large surface area/volume ratio of the Bi 2 WO 6 nanostructure promotes interaction between the sample and the bacterial membrane, potentially enhancing adsorption processes 73,78 .
According to the results, pure Bi 2 WO 6 exhibits lower antibacterial activity against both bacterial strains than nanocomposite materials of Bi 2 WO 6 /MWCNTs.Additionally, the nanocomposite proved to be more effective against S. mutans bacteria.This might be because the peptidoglycan layer of S. mutans bacteria is thinner than that of P. mirabilis bacteria, as illustrated in Fig. 12c, and because there are differences in cell physiology and metabolism 74 .Additionally, it was discovered that nanocomposite can harm bacteria's cell walls structurally and morphologically.Furthermore, the interaction between nanocomposite and bacteria's surface leads to the formation of reactive oxygen species (ROS), which can put the bacteria under oxidative stress and cause the leakage of mitochondrial enzymes and proteins that are essential for cell cycle maintenance.The increased charge transfer through the Bi-O-C bond generated by the functional groups (-OH) and (-COOH) of functionalized F-MWCNTs leading to oxygenation of the membrane as seen in FTIR spectrum analysis was also proposed as a potential mechanism.In general, nanoflake-shaped particles of Bi 2 WO 6 combined with MWCNTs have smaller average particle sizes (0.997 μm) as compared with pure Bi 2 WO 6 due to F-MWCNTs exhibiting a considerable number of defects which increases oxygen vacancies and causes the creation of ROS.This causes oxidative stress in the cell by producing (e-h) Paris through a chemical process 75 .The antimicrobial activity of MWCNTs nanocomposite is possibly due to their small size which provides a larger surface area to assist the microbial membrane damage.Further, CNTs induce oxidative stress which plays a further role in antimicrobial mechanisms 79 .Haung et al. examined the mechanical effects that influenced the antimicrobial properties of MWCNTs, such as low wear rates, low friction coefficients, favorable tribological characteristics, and high corrosion resistance 80 .Chen et al.
show that the MWCNTs played an important role as "nanodarts" which penetrated bacterial cell walls, reduced membrane potential, caused the release of genetic materials (DNA and RNA), and finally damaged the bacterial cell wall membrane 81 .The stand-out property sp 2 of carbon-based nanostructures like MWCNTs, reveals amazing electronic structures that result in semiconductivity and metallic of MWCNTs.Besides, the previous work reveals that the metallic property of MWCNTs results in higher antibacterial activity related to the electronic effect as investigated by Vecitis et.al. 82.However, several mechanisms could explain how CNTs act as an antibacterial agent such as an increase in ROS generation, attachment of MWCNTs on the microbial cell surface to stimulate the transmembrane electron transfer and induce cell wall and membrane damage, protein dysfunction, and DNA damage when MWCNTs penetrating bacterial cells 83 .
The SEM images as shown in Fig. 13A-C, have been acquired to observe the cell deformation upon interaction with pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs, respectively, and the degradation integrity of cells as well as displayed www.nature.com/scientificreports/how S.mutans and P. mirabilis membranes change after being exposed to samples after 24 h of treatment.In Fig. 13A, both untreated (control) cells displayed the typical prokaryotic cell size and intact and independent cell structures.The gram-positive S. mutans, with coccus-shaped bacteria, typically appear in clusters but they can also be found in singles or combined.While the gram-negative P. mirabilis colonies of rod-shaped with complete cell wells were validated by SEM image as can be seen in Fig. 13A.In the state of cells exposed to pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs, respectively, the SEM images display altered cell morphology that resulted in the flattening and deformation of cell membranes in Fig. 13B,C.Moreover, some of the cells exhibited morphological changes as a result of treatment with pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs, respectively.A few of the treated cells were thinner, slightly smaller in size, and less filled than the untreated cells.Numerous MWCNTs were frequently shown to aggregate with bacterial cells.According to certain investigations, MWCNTs' antibacterial properties depended on having direct contact with cells.These results suggest that after treatment with nanostructures, the cells were cruelly deforming and parts of bacterial covering and intracellular content were missing 19 .

Bacterial biofilm inhibition
Figure 14 shows how pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposites are capable of preventing P. mirabilis and S. mutans from forming biofilms.The creation of biofilms was an essential step in the start of any infection.Bacterial strain adherence to a surface, which occurs through both specific and nonspecific cell-surface interactions, is a necessary first stage in the creation of biofilms.By using a crystal violet stain on the adhering bacterial cells, these biofilms can be identified.Figure 14 illustrates how well pure Bi 2 WO 6 and Bi 2 WO 6 MWCNTs performed in this test in terms of biofilm production.According to the findings, the growth of microbiological strains was considerably inhibited by the pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite.The mechanism behind the production of reactive oxygen species (ROS) may be associated with these decreases in biofilm development.These ROS can cause lipids and proteins to peroxide and oxidize, weakening the Gram + ve cell membrane, changing fluid permeability and ion transport, and inhibiting metabolic processes 68 .Furthermore, the cell walls of Gram-positive and Gram-negative bacteria may be disrupted by physical interactions between the cell and the NPs, whether they are direct or electrostatic.
Dot plots of the S. mutans and P. mirabilis biofilms assessed by flow cytometry are shown in Fig. 15.Using excitation/emission fluorescence Syto 9 and propidium iodide stains, this assay enabled the differentiation between live and dead cell populations.It was utilized to evaluate metabolic activity in the S. mutans and P. mirabilis biofilm formed for 48 h.The percentage of live S. mutans in the untreated control bacterial strain, as shown in (Fig. 15 upper panel), was 98.2%.similarly, the percentage of live P. mirabilis was 97.2%.After treatment with Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite at a concentration of 125 μg/mL, this percentage decreases to 70.6% and 70.62% when the bacterial strain is treated with pure Bi 2 WO 6 as in (Fig. 15 middle panel).The percentage of live cells was 38.36% and 27.24% when the bacterial strains were treated with Bi 2 WO 6 /MWCNTs nanocomposite, as shown in (Fig. 15 lower panel).When it came to the reduction of living cells, the Bi 2 WO 6 / MWCNTs nanocomposite performed better than pure Bi 2 WO 6 .
Anticancer activity of pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite against liver cancer cells After 24, and 48 h of treatment with Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite, liver cancer cells were subjected to an investigation to determine the ability of Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite to inhibit growth inhibition and proliferation.This was done to investigate the inhibitory impact of Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite in comparison to the control untreated cells, Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite suppressed cell viability in a time-dependent manner, as shown in Fig. 16.The cytotoxicity effect of Bi 2 WO 6 /MWCNTs nanocomposite against normal cell line (REF) cells showed a low percent of dead cells as indicated in Fig. 16C.As a result of this study suggested that the pure Bi 2 WO 6 and the Bi 2 WO 6 /MWCNTs nanocomposite caused cell death.Many studies demonstrated the ability of functionalized CNTs to destroy cancer cells.To treat gastric cancer, Taghavi et al. 84 , created PEGylated SWCNTs that carried DOX and Bcl-xL-specific short hairpin RNA (shRNA).These treatments demonstrated increased gastric cancer cell death due to the synergistic therapeutic action of DOX and shRNA.By using the interaction between CD44 receptors expressed on tumor cells and cholic acid-derivatized HA (CAHA) wrapped around semiconducting single-walled carbon nanotubes (SWCNTs), Bhirde et al. designed a CNT-based DDS by self-assemble method to actively deliver anticancer drug DOX to tumor site 85 .The conjugation of cisplatin and EGF to the surface of oxidized SWCNTs by Bhirde et al. gave the nanosystem the capacity to target targets actively-the link between EGFR overexpression on tumor cells and EGF for increased cisplatin antitumor effectiveness 86 .Though there are still some issues to be resolved, we are optimistic that carbon nanotubes (CNTs) are very promising nanotools with excellent research and significant clinical potential in the treatment of a variety of types of cancer.

Pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs induce apoptosis in liver cancer cells
The nuclear morphology of the treated cells was examined using a dual staining method consisting of acridine orange and ethidium bromide.DNA damage was used as the criterion for evaluating apoptotic cells.Under the scope of this study, a look was also taken at how effective the Bi 2 WO 6 and the Bi 2 WO 6 /MWCNTs nanocomposites were.The AO-EB staining was utilized so that the various apoptotic characteristics of the nuclear changes could be investigated.After being stained with AO-EtBr, cells that had not undergone apoptosis were green in color, whereas apoptotic cells had an orange or red color as indicated in Fig. 17 (upper panel).As can be seen in Fig. 16 (upper panel), the cells that were treated with Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite had many more apoptotic cells than the control cells that had not been treated.Using flow cytometry to label the cancer cells with annexin V-FITC, the proportion of apoptotic cells was identified to confirm the present findings.According to MWCNTs nanocomposite-treated Hep-G2 cells, the percentage increased to 65%, and 79.5% respectively.One of these mechanisms is that they act as oxidative stimuli, which in turn promotes inflammation and DNA damage 87 .When cells were treated with pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite, the results of our study showed that the viability of Hep-G2 cells was significantly reduced.Bisht et al. showed evidence that high dosages of ZnO-Fe3O4 magnetic composite nanoparticle induced a cytotoxic impact in human breast cancer cell line (MDA-MB-231) but did not induce this effect in normal mouse fibroblast (NIH 3T3) 88 .

Pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs induce ROS generation in liver cancer cells
A considerable increase in the generation of reactive oxygen species was seen in cells treated with Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite.The fluorescence signal resulting from ROS was found to be higher when compared to the control untreated cells.The buildup of reactive oxygen species (ROS) in liver cancer cell lines after treatment with Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite was investigated in the current study.An increase of ROS was seen in cells that had been treated with Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite.ROS levels were measured with a DCFH-DA probe, as demonstrated in Fig. 18.When the liver cancer cells were treated with Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite, the results revealed that the level of reactive oxygen species (ROS) was enhanced.

Pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs cause mitochondrial dysfunction in liver cancer cells
Hep-G2 cells were stained with JC-1 to determine which mitochondria were healthy and which were damaged.On liver cancer cell lines, the impact of the Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite is assessed.To determine whether or not mitochondrial damage has occurred, it is known that mitochondrial membrane potential (∆Ψm) is produced by the proton pump of the electron transport chain, which is a component that is required for the production of ATP.For this reason, we additionally assessed MMP using JC-1 staining.As indicated in Fig. 19, the promotion of JC-1 monomers increased noticeably depending on the type of treatment that is given for liver cancer cell lines.According to the findings presented above, the treatment of liver cancer cells Hep-G2 is accompanied by a disruption of the oxidative balance in cancer cells as well as an impairment of protective anti-oxidative molecules.This causes cells to be subjected to excessive oxidative stress and mitochondrial dysfunction which leads to the subsequent release of cytochrome c which causes the activation of caspases-9 and caspase-3 pathways.Preparation of Bi 2 WO 6 /MWCNTs nanocomposite and evaluation of their antibacterial and anticancer activities are presented in Fig. 20.

Conclusion
In this study, pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite were successfully fabricated using a hydrothermal approach.The characterization methods demonstrated that Bi 2 WO 6 retained its orthorhombic structure and the introduction of small amounts of MWCNTs had little impact on the morphology, structure, optical, and composition of Bi 2 WO 6 flower-like structure as demonstrated using FESEM, EDX TEM, XRD, FTIR, and Raman.Besides, this configuration enables the nanocomposite to take advantage of the high adsorption capacity and large specific surface area of MWCNTs, leading to the formation of additional active sites as revealed by FESEM and TEM.The addition small amount of MWCNTs led to significant quantum confinement-induced band gap narrowing of Bi 2 WO 6 in the nanocomposite.Additionally, the Bi 2 WO 6 /MWCNTs nanocomposite's band gap energy drops to 2.6 eV, which is consistent with the light absorption range shifting to the red region and the associated reduced band gap energy.The Bi 2 WO 6 /MWCNTs nanocomposite's PL spectrum improved the rate at which carriers separated, leading to the formation of more active sites and an increase in adsorption capacity.
Moreover, pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite reveal its antibacterial effects against P. mirabilis and S. mutans bacteria and demonstrated antibacterial efficacy and biofilm formation, enhancing its properties in comparison to pure Bi 2 WO 6 .SEM analysis and biofilm test further reveal the efficiency of Bi 2 WO 6 / MWCNTs nanocomposite in cell destruction process with cell membrane envelope and spreading to the intracellular as well as demonstrated the dimension dependence of Bi 2 WO 6 /MWCNTs and enhancing its properties compared with pure Bi 2 WO 6 .Generally, the results of the current study showed that the Bi 2 WO 6 and Bi 2 WO 6 / MWCNTs nanocomposite considerably reduced bacterial biofilm formation.Also, Bi 2 WO 6 /MWCNTs nanocomposite acts as cytotoxic and apoptosis inducer in liver cancer cells via modulates ROS pathway.Furthermore, these results recommend that pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite could be promising materials for developing antimicrobial and cancer agents.

Figure 6 .
Figure 6.FESEM images of the (a,b) pure Bi 2 WO 6 nanostructures and inset image of average particle size.

Figure 8 .
Figure 8. EDS spectrum of the (a) pure Bi 2 WO 6 nanostructures and Bi 2 WO 6 /MWCNTs nanocomposite (b) inset is the table showing the percentage of each component in Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite.

Figure 11 .
Figure 11.Antibacterial activity of pure Bi 2 WO 6 nanostructure under visible against (A) P. mirabilis and (B) S. mutans.(C) Data are represented as mean ± SD of three independent experiments.

Figure 12 .
Figure 12.Antibacterial activity of Bi 2 WO 6 /MWCNTs nanocomposite against (A) P. mirabilis and (B) S. mutans.(C) Data are represented as mean ± SD of three independent experiments.

Figure 13 .
Figure 13.SEM images visualized the morphological effect of pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs on bacterial strains of S. mutans and P. mirabilis.The bacterial strains showed changes in the cell membranes like damaged, blabbed, and clumped membranes.Un-treated control bacterial strains (A).Bacterial strains treated with pure Bi 2 WO 6 (B).Bacterial strains treated with Bi 2 WO 6 /MWCNTs (C).

Figure 14 .Figure 15 .
Figure 14.Pure Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs reduce biofilm formation in bacterial strains.Data are represented as mean ± SD of three independent experiments.

Figure 20 .
Figure 20.Preparation of Bi2WO6/MWCNTs nanocomposite and evaluation of their antibacterial and anticancer activities.
4cells per well.After removing the growth medium and replacing it with 200 µL of new medium containing various concentrations of Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite at a concentration (25 µg/mL) for 24, 48 h 36 .

Table 3 .
Position and assignation of Raman bands for Bi 2 WO 6 and Bi 2 WO 6 /MWCNTs nanocomposite synthesized by hydrothermal method.